A significant proportion of therapeutic drug candidates fail to become marketable drugs because of adverse metabolism or toxicity discovered during clinical trials. These failures represent a very significant waste of development expenditure and consequently there is a need for new technologies that can more reliably, quickly and economically predict at the pre-clinical development stage the metabolic and toxicological characteristics of drug candidates in man. At present, most pre-clinical metabolic and toxicity testing of drug candidates relies on laboratory animals, human and/or mammalian cell lines and/or tissues in culture. However, none of these methods is completely reliable in predicting metabolism or toxicity in a human subject. Metabolic and toxicological data from animals can differ significantly from that obtained from a human subject due to species differences in the biochemical mechanisms involved. In addition, interpretation of data derived from in vitro human cell cultures or isolated human tissue studies can be problematic since such systems are not available for all organs and tissues or they fail to retain the same metabolic characteristics as they possess in vivo.
It is known in the prior art that the metabolism, distribution and toxicity of most drugs depends on their interactions with four distinct main classes of proteins:    a) Phase-1 drug-metabolising enzymes, such as the cytochromes P450 which generally add or expose polar groups on the xenobiotic molecule;    b) Phase-2 drug-metabolising enzymes, such as transferases, in particular the glucuronyl transferases, glutathione transferases, sulphonyl transferases and acetyl transferases which conjugate the polarised xenobiotic molecule to a hydrophilic group thereby facilitating its subsequent excretion;    c) Drug transporter proteins, such as the ATP-binding cassette proteins which include the multi-drug resistance proteins (MDRs) and multi-drug resistance-associated proteins (MRPs) and the organic anion transporting polypeptides (OATPs) which facilitate the transport of drugs and other xenobiotic molecules across plasma membranes;    d) Transcription factors, such as the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) which regulate the transcription of genes encoding proteins of the preceding classes, in particular the cytochromes P450.
Variation between species is known in each of these protein classes both with respect to the multiplicity of proteins within each class, the function of the proteins themselves and with respect to genetic regulation of their expression.
It is known from WO2004/007708 how to produce non-human transgenic animals expressing functioning human P450s in which the functions of endogenous cytochromes P450 have been annulled by deletion of individual P450 genes or by deletion of the cytochrome P450 reductase gene encoding the enzyme on which the function of all cytochromes P450 depends. However, the described animal model has limitations in that not only are the introduced P450s restricted to particular organs or tissues of the non-human animal but also there is no provision to regulate expression of the human P450s in a manner that is analogous to that seen in the human. One of the prior art models is also limited in that additional modifications are needed to provide cytochrome P450 reductase activity to the introduced human P450s without reactivating endogenous non-human P450s. A yet further disadvantage resides in the lack of provision to reproduce human phase-2 metabolism, thus the system is unable to provide an entire metabolic profile.
It is also known from the prior art to humanise the induction characteristics of cytochromes P450 in the mouse by expressing human PXR (Xie et al, Nature Vol 406, 435-9, 2000) or human CAR (Zhang et al, Science Vol 298, 422-4, 2002) in a mouse wherein the mouse PXR gene and/or mouse CAR gene respectively have been deleted. While such animals demonstrate induction patterns of endogenous P450s that reproduce those seen in the human they have undesirable characteristics because the cytochromes P450 whose expression is regulated analogously to the human are still non-human cytochromes P450. A further disadvantage is that because the PXR or CAR genes themselves are not regulated as they are in the human by virtue of the transgene being driven by a heterologous tissue-specific promoter (albumin promoter), over-expression of the heterologous gene can occur which can have the result that a normal metabolic pathway is bypassed. Moreover, the PXR and CAR transgenes are derived from a cDNA rather than a genomic clone, thus the transgenic non-human animals consequently lack the sequences necessary correctly to reproduce all the transcriptional and post-transcriptional regulation of PXR or CAR expression hence their expression is restricted to the liver and may not be of a physiological level. In addition these models do not encode for splice variants of the human gene. Another drawback of the PXR/CAR models is that they are unsuitable to combine with modifications of other genes within one animal since the humanisation of each gene is achieved by two independent genomic alterations: (i) knock-out of the endogenous gene (ii) transgenesis with the human orthologue under control of the albumin promoter at a different genomic location.
Ma et al. (Drug Metab Dispos. 2007 February;35(2):194-200) introduced the complete human PXR gene, including 5′ and 3′ flanking sequences, into PXR knock-out mice by bacterial artificial chromosome (BAC) transgenesis. They observed selective expression of human PXR in the liver and intestine. Treatment of PXR-humanised mice with PXR ligands mimicked the human response, as both hepatic and intestinal Cyp3a11 mRNA and protein were strongly induced by rifampicin, a human-specific PXR ligand, but not by pregnenolone 16α-carbonitrile (PCN), a rodent-specific PXR ligand. In wild-type mice, Cyp3a11 mRNA was strongly induced by PCN, but not by rifampicin.
There is therefore a need for improvements in animal models of human metabolism that can control expression of the human genes introduced into the animal so that their expression is regulated in a manner more closely analogous to that seen in humans. There is also a need for more aspects of the human metabolic pathway to be reproduced. Effective animal models of human metabolism require not only expression of the relevant human proteins but also annulment of the functions of the homologous endogenous proteins.
One reason why the present invention embodies a surprising advance over the prior art is that many prior art researchers appear to be of the view that the problem posed by the need for models of drug metabolism is already solved. For example, Xie and Evans (2002, DDT7, p 509) state that humanising PXR is “one of the rare examples where replacing a single transcriptional regulator allows conversion of species-specific gene regulation”. Furthermore, it is evident that workers have turned their attention to techniques that differ markedly to those that utilise transgenic animal systems. For example, attempts are being made to humanise the mouse liver as an organ by using human hepatocytes, the aim being to obtain a mouse model for drug metabolism in humans. This work is labour-intensive though, and in the inventors' opinion is of dubious relevance to the situation in reality.
The present invention is the first methodology that takes into account all of the problems that prior art systems suffer from and that seeks to resolve these problems in a practical manner. The inventors have recognised that in order to provide transgenic non-human animal models with humanised drug metabolism pathways that overcome the undesirable features of the animal models described in the prior art a number of criteria should ideally be satisfied:    a) Regulation of the expression of introduced human proteins such that patterns of expression in the human are reproduced;    b) Expression of multiple human proteins so that multiple aspects of human metabolism are reproduced;    c) Annulled expression or function of multiple endogenous genes so that interference from non-human metabolic pathways on the functions of introduced human proteins is significantly reduced.
In the present invention, we provide methods of producing non-human animal cell and non-human transgenic animals that incorporate at least some if not all of these desired qualities. Such non-human animal cell and non-human transgenic animals possess desirable characteristics not available in the prior art in that they can model entire human pathways of xenobiotic metabolism rather than just individual elements of pathways and that such models are provided for all tissues and organs. This is achieved through the application of technical approaches hitherto not available in the prior art with respect to obtaining regulation of transgene expression analogous to that seen in human cells through the use of extensive regulatory DNA sequences and with respect to annulment of endogenous metabolic pathways through deletion or gene exchange. A number of relevant human proteins are expressed in a single animal.